Method for culturing bacillus subtilis, cultured microorganism obtained by the method, water-soluble vitamin K derivative originating in the cultured microorganism, and food product, beverage, or feed containing the cultured microorganism or the vitamin K derivative

ABSTRACT

A method for culturing  Bacillus subtilis  in such a manner that a vitamin K derivative is accumulated in the largest amount in the cells of the microorganism. A method for the culture of  Bacillus subtilis , which comprises culturing  Bacillus subtilis  and recovering the cells of the microorganism before the vitamin K produced within the cells of the microorganism is released from the cells is disclosed, as well as the cultured product of  Bacillus subtilis  cultured by this method, a water-soluble vitamin K derivative originating in the cultured product, a food product, beverage, or feed containing the cultured product and/or the water-soluble vitamin K derivative, and a method for extracting vitamin K.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a method for culturing Bacillussubtilis, the cultured microorganism obtained by the method, awater-soluble vitamin K derivative originating in the culturedmicroorganism, and a food product, beverage, or feed containing thecultured microorganism or the water-soluble vitamin K derivative. Moreparticularly, this invention relates to a method for culturing Bacillussubtilis in such a manner as to induce storage of a vitamin Kderivative, particularly a menaquinone-7 (vitamin K₂) derivative, in thelargest amount within the cells of the Bacillus subtilis, a culturedproduct of Bacillus subtilis having a vitamin K derivative, particularlya menaquinone-7 (vitamin K₂) derivative, accumulated in a large amountwithin the cells thereof in consequence of the culture by the methodmentioned above, a water-soluble vitamin K derivative, particularly awater-soluble menaquinone-7 (vitamin K₂) derivative, originating in thecultured product mentioned above, and a food product, beverage, or feedcontaining the cultured product or the water-soluble vitamin Kderivative, particularly the water-soluble menaquinone-7 (vitamin K₂)derivative.

[0003] 2. Description of the Related Art

[0004] Vitamin K has been heretofore known as a factor found necessaryduring the coagulation of blood. Since the deficiency of this vitaminbrings deterioration of the ability to coagulate blood, vitamin K isheld to constitute itself one species of fat-soluble vitamin otherwisecalled an antihemorrhagic vitamin. As regards the cause for thedeterioration of ability to coagulate blood thus brought about by thedeficiency in this vitamin K, it has been suggested in recent years thatvitamin K is indispensable for the biosynthesis of several bloodcoagulation factors including prothrombin. The amount of the vitamin Kwhich is found necessary for the purpose of preventing the deteriorationof the blood-coagulating ability is extremely small, i.e. on the orderof μg. Generally, since adults are supplied with this vitamin K byenterobacteria, they rarely contract the disease of vitamin Kdeficiency. The synthetic vitamins K₁ and K₂ are used as medicines forthe therapy of the disease of the vitamin K deficiency hemorrhage. Todate, the vitamin K has not attracted much attention because theconcentrate of natural vitamin K₁ has been utilized heretofore in theform of a food product for the purpose of preventing this disease.

[0005] It has been demonstrated in recent years, however, that vitamin Khas a function of promoting ossification and a function of repressingbone resorption and that the density of bone is increased by theadministration of the vitamin K. The osteoporosis is the pathosis ofembrittlement of bone caused by senility or illness. Since this diseaseaccompanies fracture and severe pain, it has been posing a serioussocial issue from the standpoint of geriatric therapy. A study conductedon patients with osteoporosis to determine their vitamin K levels inblood has demonstrated that the vitamin K levels average about one halfof those of healthy persons. As a prospective medicine for the treatmentof osteoporosis, therefore, the synthetic vitamin K is now under aclinical test. It has been established by the clinical test that in thetreatment and prevention of osteoporosis, unlike the case of treatingand preventing hemorrhage, the administration of vitamin K in such alarge daily dose of not less than 45 mg is effective in increasing theamount of bone. The osteoporosis favors the prevention thereof ratherthan the treatment to be effected after the onset of the disease. Forthe sake of this prevention, daily ingestion of the vitamin K from foodis commendable. The ingestion of the vitamin K in the dose mentionedabove from the existing food seems to be difficult, however, because thequestion how much of the vitamin K ought to be ingested daily toincrease the amount of bone and attain the prevention of osteoporosisremains yet to be elucidated.

[0006] The ingestion of vitamin K is preferred to be attained by thedaily food as described above. In fact, vitamin K₁ can be taken fromgreenish yellow vegetables and marine plants and vitamin K₂ from suchfermented food products as fermented soybeans (natto). An effort toattain the ingestion of 45 mg of the vitamin K, i.e. an amount reportedto be effective in improving osteoporosis, from the commerciallyavailable food product, however, actually turns out to be a verydifficult task as aptly evinced by the fact that a food productcontaining 1 ppm of vitamin K, for example, ought to be consumed in sucha large amount as 45 kg daily to fulfill the ingestion under discussion.It is natto, among other food products, that has the largest vitamin Kcontent on the order of ten-odd ppm. Even the natto so renowned ought tobe consumed in such a large amount as some hundreds of g to several kgdaily to fulfill the ingestion. From the standpoint of taste, it isdifficult for any person to form the habit of eating the natto in such alarge amount as mentioned above daily. In addition to this difficulty,the ingested vitamin K has a short half-life period. The vitamin K hassuch unsolved problems as failing to manifest the effect thereof fullysatisfactory when it is orally ingested independently and tending toentail adverse reactions when it is ingested in an unduly large amountall at once. While the ingestion of the vitamin K in the concentratedform is commendable, the commercially available natural vitamin Kconcentrate which additionally incorporates therein prepared milk powderfor the sake of preventing hemorrhage is expensive and the syntheticvitamin K offered as a pharmaceutical preparation is not usable as food.

[0007] Incidentally, in the class of vitamin K's, it are only thevitamin K₁ and K₂ groups that occur in nature. The vitamin K₁ iscopiously contained particularly in green vegetables, vegetable oils,and marine products among other kinds of food. Seaweed, laver, and tealeaves, for example, contain some tens of ppm of vitamin K₁ and soybeanoil, spinach, and broccoli, for example, contain several ppm of vitaminK₁. This vitamin K₁ is synthesized by condensing2-methyl-1,4-naphthoquinone and phytyl acetate. Further, the vitamin K₂group is known as varying homologs of menaquinone-l to -14 (MK-1 toMK-14), depending on the difference in chain length. Among otherhomologs, particularly the menaquinone-7 (occasionally referred tosimply as “MK-7” in the present specification) is a typical substance ofthe vitamin K₂ and is synthesized mainly by Bacillus subtilis natto. Innature, the MK-7 is isolated only with unusual difficulty because itoccurs in a relatively minute amount in the range of several to ten-oddppm even in natto and has a short half-life period. So far, theinvention of JP-A-08-73,396 has been known as the sole case ofsucceeding in preparing a lipid having a high MK-7 content.

[0008] Thus, quantity production of vitamin K2 by the use of such amicroorganism as Bacillus subtilis natto has been attempted. Manystudies have been known to have perfected methods for producing naturalvitamin K₂. Methods for collecting vitamin K₂ from the culture broth ofa microorganism belonging to genus Flavobacterium (JP-B-07-28,748 andJP-B-07-51,070) and methods for producing vitamin K by inoculatingBacillus subtilis natto -to soy beam soup stock or soy-bean cake leesand fermenting the microorganism in the medium (JP-A-10-295,393,JP-A-08-19,378, JP-A-08-9,916, and JP-A-08-173,078) may be cited asexamples of the outcomes of such studies. In addition to these methods,a method for obtaining a concentrated lipid containing natural vitaminK₂, particularly natural MK-7, in a large amount by subjecting thefermented cells of Bacillus subtilis natto to extraction with such anorganic solvent as alcohol, ether, ester, or ketone has been proposed(JP-A-08-73,396). The methods which use such a vitamin K-producingmicroorganism as Flavobacterium have the problem that the vitamin K₂obtained thereby can not be utilized in its unmodified form for a foodproduct because the safety of Flavobacterium as food has not beenestablished. Though the methods which prepare the vitamin K by the useof Bacillus subtilis natto indeed obtain cultures with relatively highvitamin K contents reaching the maximum of about 40 mg/litter of culturebroth, the products thereof find utility only in heavily restrictedapplications because they are not water-soluble but fat-soluble bulks ofvitamin K. Further, the lipid with a high natural menaquinone-7 contentprepared by extracting the fermented cells of Bacillus subtilis nattowith an organic solvent, despite the use of such raw materials as soybeans which are available for food, uses the organic solvent and,'therefore, requires thorough removal of the organic solvent before itis used in food. This removal of the organic solvent necessitatesprovision of a special device intended exclusively therefor and entailsan addition to the time required for the operation. The lipid of highMK-7 content to be obtained is a fat-soluble product, similarly to theproducts mentioned above, as clearly inferred from the designationthereof and, consequently, finds utility only in limited applications.

[0009] Many methods for producing vitamin K and MK-7 by using suchmicroorganisms as Bacillus subtilis natto from the fermented broths ofnatto and such by-products as lees generated during the course ofmanufacture of natto and soup stock (namely for extracting vitamin K andMK-7 from the cells of the microorganism) have been reported inliterature. Virtually no reports have been heretofore made concerningvitamin K and MK-7 stored within the cells of species of Bacillussubtilis represented by Bacillus subtilis natto.

[0010] An object of this invention, therefore, is to provide a methodfor culturing Bacillus subtilis in such a manner as to induce storage ofa vitamin K derivative, particularly a menaquinone-7 (vitamin K₂)derivative, in the largest amount within the cells of the Bacillussubtilis, a cultured product of Bacillus subtilis having a vitamin Kderivative, particularly a menaquinone-7 (vitamin K₂) derivative,accumulated in a large amount within the cells thereof in consequence ofthe culture by the method mentioned above, a water-soluble vitamin Kderivative, particularly a water-soluble menaquinone-7 (vitamin K₂)derivative, originating in the cultured product mentioned above, and afood product, beverage, or feed containing the cultured productmentioned above or the water-soluble vitamin K derivative, particularlythe water-soluble menaquinone-7 (vitamin K₂) derivative.

[0011] Another object of this invention, directed at rendering naturalvitamin K, particularly natural MK-7, which either cannot be ingested atall or may be ingested only with difficulty, in a sufficient amount fromthe ordinary food product ingestible easily and daily, is to provide amethod for culturing Bacillus subtilis in such a manner as to inducestorage of a vitamin K derivative, particularly a menaquinone-7 (vitaminK₂) derivative, in the largest amount within the cells of the Bacillussubtilis, a cultured product of Bacillus subtilis having a vitamin Kderivative, particularly a menaquinone-7 (vitamin K₂) derivative,accumulated in a large amount within the cells thereof in consequence ofthe culture by the method mentioned above, a water-soluble vitamin Kderivative, particularly a water-soluble menaquinone-7 (vitamin K₂)derivative, originating in the cultured product mentioned above, and afood product, beverage, or feed containing the cultured productmentioned above or the water-soluble vitamin K derivative, particularlythe water-soluble menaquinone-7 (vitamin K₂) derivative.

[0012] Still another object of this invention, directed at renderingnatural vitamin K, particularly natural MK-7, which either cannot beingested at all or may be ingested only with difficulty, in a sufficientamount from the ordinary food product ingestible easily and daily, is toprovide a method for culturing Bacillus subtilis in such a manner as toinduce storage of a vitamin K derivative, particularly a menaquinone-7(vitamin K2) derivative, in the largest amount within the cells of theBacillus subtilis, a cultured product of Bacillus subtilis having avitamin K derivative, particularly a menaquinone-7 (vitamin K₂)derivative, having effects of maintaining the level thereof in blood asheightened to a necessary level for a long time and at the same timeexcelling in safety accumulated in a large amount within the cellsthereof in consequence of the culture by the method mentioned above, awater-soluble vitamin K derivative, particularly a water-solublemenaquinone-7 (vitamin K₂) derivative, originating in the culturedproduct mentioned above, and a food product, beverage, or feedcontaining the cultured product mentioned above or the water-solublevitamin K derivative, particularly the water-soluble menaquinone-7(vitamin K₂) derivative.

SUMMARY OF THE INVENTION

[0013] The present inventor, in appreciation of the true state of priorart mentioned above, has pursued a diligent study on the components ofnatto (Experientia, 43:1110, 1987; Acta Haematol., 84: 139, 1990;Fibrinolysis, 6: 86, 1992; Journal of Japan Society of Pharmacists, 30:73, 1994; Bio-Industry, 14: 47, 1996) or the analysis of vitamin K inBacillus subtilis natto and in blood (Japan Thrombotic HemostasisJournal, 8: 287, 1997; Fibrinolysis & Proteolysis, Vol. 12, Supplement1, 205, p. 75,-1998) and has consequently found that rather than theingestion of the fermented product of Bacillus subtilis natto such asfermented soybeans or the vitamin K contained therein, the ingestion oflive Bacillus subtilis itself is recognized to be highly effective inpromoting the level of vitamin K, particularly MK-7, in plasma and thatparticularly the ingestion of Bacillus subtilis natto manifests effectsof maintaining the level in plasma at an extremely high level ascompared with the ingestion of other substances.

[0014] The present inventor has also found that Bacillus subtilis nattocultured to a specific stage of growth has MK-7 accumulated in a largeamount in the cells thereof, that by ingesting the Bacillus subtilisnatto or the fermented product thereof recovered at this stage, it isrecognized to heighten the level of vitamin K, particularly MK-7, inplasma, and that the level of the vitamin K in plasma obtained inconsequence of the ingestion is maintained for a long time. In additionto this knowledge, the inventor has also found that the vitamin K,particularly the MK-7, accumulated in the cells of Bacillus subtilisnatto recovered at the stage mentioned above is soluble in water and hascome to entertain expectations that applications of the water-solublevitamin K, particularly MK-7, will greatly expand.

[0015] This invention has been perfected based on the knowledge asdescribed above.

[0016] Specifically, the objects mentioned above can be accomplished bya method for culturing Bacillus subtilis, comprising the steps ofculturing Bacillus subtilis and recovering the cells of said Bacillussubtilis before the vitamin K produced in the cells is released from thecells.

[0017] The objects mentioned above can be further accomplished by acultured product of Bacillus subtilis obtained by the culturing methodof this invention, a water-soluble vitamin K derivative originating inthe cultured product of Bacillus subtilis, and a food product, beverage,or feed containing the fermented product mentioned above and/or thewater-soluble vitamin K derivative.

[0018] The method for culturing Bacillus subtilis according to thisinvention is characterized by culturing Bacillus subtilis and recoveringthe cells of the Bacillus subtilis before the vitamin K produced in thecells is released from the cells. By the method of this invention,therefore, the Bacillus subtilis used therein can be recovered in astate having the vitamin K derivative, particular the menaquinone-7derivative, stored in the largest amount within the cells thereof. Forthis reason, the water-soluble vitamin K, particularly the menaquinone-7derivative, can be recovered in a larger amount from the culturedproduct as compared with the conventional method. Further, particularlywhen Bacillus subtilis natto is used, since the safety thereof has beenensured, the cultured product obtained by the method mentioned above orthe food product, beverage, or feed containing the water-soluble vitaminK derivative originating in the cultured product contains thewater-soluble vitamin K derivative, particularly the menaquinone-7derivative, in a larger amount and possesses higher safety as comparedwith the conventional product. The practice of eating this food product,beverage, or feed can be expected to ensure efficiently simple dailyingestion of vitamin K and advance further the improvement ofosteoporosis.

[0019] The water-soluble vitamin K derivative originating in thecultured product obtained by the method of the present inventionembraces an epochal invention purporting that the vitamin K heretoforeobtained in a fat-soluble form can be converted into a water-solublequality. Further, the water-soluble vitamin K derivative has excellentphotostability. This particular invention can be expected not only toenlarge appreciably the use found for the new vitamin K as compared withthe conventional fat-soluble vitamin K but also to confer a very highvalue on this invention from the industrial point of view.

[0020] Further, since the practice of ingesting the food product,beverage, or feed containing the cultured product or the water-solublevitamin K derivative can be expected to allow more effective preventionof osteoporosis because it permits the level of vitamin K in plasma tobe efficiently heightened and enables the heightened level in plasma tolast for a far longer period than the vitamin K assimilated fromconventional medicines or food products.

[0021] The above and other objects, features and advantages of thepresent invention will become clear from the following description ofthe preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a graph showing time-course changes in levels of vitaminK (menaquinone-7) in plasma of five healthy adults each having orallyingested a dose of 1 g of dry Bacillus subtilis natto prepared inExample 1.

[0023]FIG. 2 is a graph showing time-course changes in levels of vitaminK (manequinone-7) in plasma of five healthy adults each having orallyingested a dose of 1.8×10¹⁰ cells of dry Bacillus subtilis nattoprepared in Example 2.

[0024]FIG. 3 is a graph showing the growth of various species ofBacillus subtilis natto relative to the time of culture in Example 3.

[0025]FIG. 4 is a graph showing the amounts of MK-7 in the culturedbroths of various species of Bacillus subtilis natto relative to thetime of culture in Example 3.

[0026]FIG. 5 is a graph showing the amounts of MK-7 accumulated in thecells of various species of Bacillus subtilis natto relative to the timeof culture in Example 3.

[0027]FIG. 6 is a graph showing the amounts of MK-7 accumulated in thecells, the fibrinolytic activity, and the amount of MK-7 released fromthe cells in Example 4.

[0028]FIG. 7 is a graph showing the amounts of MK-7 relative to the timeof exposure to the light of a fluorescent lamp in Example 7 andComparative Example 1.

[0029]FIG. 8 is a graph showing the patterns during gel filtration andthe amount of MK-7 in each fraction in Example 10.

[0030]FIG. 9 is a graph showing the results of the HPLC analysisperformed on a water-soluble vitamin K derivative and an isopropanolextract of tofu (bean curd) refuse in Example 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

[0031] This invention will be described in detail below.

[0032] According to the first aspect, this invention provides a methodfor culturing Bacillus subtilis, which comprises culturing Bacillussubtilis and recovering the cells of said Bacillus subtilis before thevitamin K produced in the cells is released from the cells.

[0033] The Bacillus subtilis to be used in this invention does not needto impose any restriction particularly but requires only to belong togenus Bacillus subtilis and possess the ability to produce vitamin K.Any of the known species of genus Bacillus subtilis can be used.Particularly in consideration of such factors as safety and amount ofvitamin K to be produced, Bacillus subtilis natto may be preferably usedin this invention. Bacillus subtilis natto preferably used in thisinvention does not need to impose any restriction particularly. Astypical examples thereof, such commercially available species ofBacillus subtilis natto as Takahashi strain (made by Yuzo TakahashiLaboratory in Yamagata), Naruse strain (made by Naruse FermentationChemical Laboratory K. K. in Tokyo), Miyagino strain (made by MiyaginoNatto Manufactory K.K. in Sendai), Asahi strain (made by Asahi KogyoK.K. in Tokyo), Nitto strain (made by Nitto Yakuhin Kogyo K.K. inKyoto), and Meguro strain (made by Meguro Laboratory K.K. in Osaka) andUnnan SL-001 strain may be cited. In consideration of the amount ofvitamin K derivative to be accumulated in the cells, Meguro strain,Miyagino strain, Takahashi strain, Naruse strain, Asahi strain, Nittostrain, and Unnan SL-001 strain may be used in the order of decreasingpreferability (the amounts of vitamin K derivative produced increase inthis order). Incidentally, Unnan SL-001 strain was internationallydeposited under FERM BP-6713 at the National Institute Bioscience andHuman-Technology Agency of Industrial Science and Technology on May 7,1999.

[0034] In this invention, it is essential that the culture of Bacillussubtilis be terminated before the vitamin K produced in the microorganiccells is released from the cells and the vitamin K be recovered from thecells. For the culture of Bacillus subtilis as contemplated by thisinvention (with respect to composition of culture medium and cultureconditions), the same composition of culture medium and cultureconditions as are generally employed in the culture of any species ofBacillus subtilis can be used. Since this culture permits selective andcopious accumulation of the vitamin K in the microorganic cells withoutbeing released in any appreciable amount from the cells, the cellscontaining the vitamin K in a large amount therein may be obtained by asimple procedure of recovering the cells in the specific state mentionedabove. The cells thus recovered, particularly when the microorganism hasthe safety thereof established as in the case of Bacillus subtilisnatto, can be used for the purpose of ingestion in the unmodified formwithout undergoing such an extra work as of extracting the vitamin Kthereof, alternatively the cultured cells can be incorporated in anordinary food product.

[0035] In this invention, the time for recovering the cells of Bacillussubtilis must precede the release of the produced vitamin K from thecells as mentioned above. Preferably, it may be the time at which thenumber of microorganic cells shifts from the logarithmic growth phase tothe maximum stationary phase. Particularly when Bacillus subtilis isBacillus subtilis natto, the recovery of the cells may be preferablymade when the logarithmic growth phase is in the process of shifting tothe maximum stationary phase and the production of nattokinase has notbeen started. More specifically, though the culturing time is variablewith such culturing conditions as culturing temperature, pH, and initialconcentration of cells and the culturing method as well, when UnnanSL-001 strain is inoculated at an initial concentration of 5×10⁶/ml to300 ml of a culture broth (containing 1.5% polypeptone-S, 1% glucose,and 0.1% yeast extract, pH 7.2) in an Erlenmeyer flask having an innervolume of 500 ml and shaking-cultured therein (100 rpm) at 37° C., forexample, the time falls between 0.5 and four days and preferably occursimmediately after the absorbance at 660 nm reaches the maximum level(one to two days thereafter).

[0036] In this invention, for the culture of Bacillus subtilis, the sameculture medium and culture conditions as are generally adopted forculturing any species of Bacillus subtilis may be used. For example, theculture medium to be used for the culture which is contemplated by thisinvention does not need to be restricted particularly. Any of theculture media using such components as are well-known to persons skilledin the art can be used. A culture medium prepared by suitably mixingvarious culture components, a commercially available culture medium inits unmodified form, or a culture medium obtained by having theaforementioned known components additionally incorporated in thecommercially available culture medium may be used. The culture mediumthus used may be either a solid medium or a liquid medium, whicheverbetter suits the purpose for which the medium is to be used. The culturemedium is only required to contain a carbon source, a nitrogen source ina proper amount, an inorganic salt, and other nutrients which can beassimilated by the microorganism to be used, without reference to thechoice between a synthetic medium and a natural medium.

[0037] The carbon source which can be used in the culture of Bacillussubtilis according to this invention is variable with the species of themicroorganism to be used. It imposes no restriction particularly butrequires only to allow the strain in use to grow satisfactorily andproduce vitamin K with high efficiency. As typical examples of thecarbon source, starch and compositional fractions thereof, roasteddextrin, processed starch, starch derivative, physically treated starch,α-starch, soluble starch, amylose, amylopectin, malt oligo sugar,oligosaccharide, cyclodextrin, pullulan, corn starch, potato starch,sweet potato starch and dextrin, glycerin, sorbitol, malt extract, andglucose and other similar carbohydrates may be cited. From the viewpointof the production of vitamin K, glucose and starch may be preferablyused among other carbon sources mentioned above. These carbon sourcesmay be used either singly or in the form of a mixture of two or moremembers.

[0038] The nitrogen source to be used in the culture of Bacillussubtilis according to this invention is likewise variable with thespecies of the microorganism to be used. It imposes no restrictionparticularly but requires only to allow the strain in use to growsatisfactorily and produce vitamin K with high efficiency. As typicalexamples of the nitrogen source, meat extract, malt extract, peptone,polypeptone derived from soybean (such as, for example, polypeptone-S),yeast extract, tasty liquid (acid hydrolyzate of soy bean starch),soybean powder, milk casein, Casamino acid, organic nitrogen compoundssuch as various amino acids and corn steep liquor, ammonia, ammoniumsalts such as ammonium nitrate, ammonium sulfate, and ammonium chloride,nitrates such as sodium nitrate, and inorganic nitrogen compounds suchas urea may be cited. From the viewpoint of the production of vitamin K,polypeptone derived from soybean (such as, for example, polypeptone-S)and soybean powder are preferably used among other nitrogen sourcesmentioned above. These nitrogen sources can be used either singly or inthe form of a mixture of two or more members.

[0039] The inorganic salt to be used in the culture of Bacillus subtilisaccording to this invention is likewise variable with the species of themicroorganism to be used. It imposes no restriction particularly butrequires only to allow the strain in use to grow satisfactorily andproduce vitamin K with high efficiency. As typical examples of theinorganic salt, phosphates, hydrochlorides, sulfates, acetates, and etc.of magnesium, manganese, calcium, sodium, potassium, copper, iron, andzinc may be cited. These inorganic salts can be used either singly or inthe form of a mixture of two or more members.

[0040] The commercially available culture media which can be used forthe culture according to this invention include nutrient broth (drybouillon) (produced by Nissui Seiyaku K.K. or Nippon Seiyaku K.K.) andpolypeptone-S (made by Wako Pure Chemical Industries, Ltd.), forexample.

[0041] Alternatively, in this invention, as the culture medium for usein the culture of Bacillus subtilis contemplated thereby, such materialsas tofu (bean-curd) refuse, soybean, soybean soup stock by-producedduring the manufacture of soybean paste and fermented soybeans (natto),soybean cake lees by-produced during the manufacture of soybean cake anddeep-fried soybean cake, soybean cake lees by-produced during themanufacture of edible oil from soybean as the raw material, and soybeanseed rind by-produced during the manufacture of soybean paste which canbe fermented by Bacillus subtilis natto can be used. In this case, theculture medium, when necessary, may suitably incorporate therein suchcarbon sources, nitrogen sources, and inorganic salts as mentionedabove.

[0042] In this invention, the culture of Bacillus subtilis may becarried out in the same manner as in the method heretofore known in theart. The culture conditions used according to this invention can beproperly selected to suit the microorganic strain, the composition ofculture medium, and the method of culture to be adopted. They impose norestriction particularly but require only to allow the microorganicstrain in use to propagate and produce vitamin K with high efficiency.The culturing temperature may be generally in the range of 20° to 45 °C., preferably in the range of 370 to 42° C. and the pH value of theculture medium proper for the culture may be generally in the range of6.0 to 9.5, preferably in the range of 7.0 to 8.5.

[0043] According to the second aspect, this invention provides thecultured product of Bacillus subtilis obtained by the method describedabove.

[0044] In this invention, the phrase “cultured product of Bacillussubtilis” may include both the cultured cell of Bacillus subtilis andthe product yielded out the cell of Bacillus subtilis. In latter case,Unnan SL-001 strain may be preferably used because higher yield out thecell thereof.

[0045] According to the third aspect, this invention provides thewater-soluble vitamin K derivative which originates in the culturedproduct according to the second aspect of this invention.

[0046] The cultured product of Bacillus subtilis obtained by the methodof this invention may have a large amount of vitamin K, particularlyvitamin K₂, and more particularly menaquinone-7 (MK-7), accumulated inthe cells thereof as described above. Specifically, the amounts ofvitamin K, vitamin K₂, and MK-7 accumulated in the cultured product ofBacillus subtilis according to the method of this invention, thoughvariable with the used strain, the kind of a culture medium, and theculturing conditions to be adopted, generally fall each in the range of10 to 200 mg/100 g of vacuum dried cells. More specifically, when Megurostrain is shaking-cultured (100 rpm) in a medium containing 3% ofsoybean peptone at 37° C. for two days, the maximum amount of MK-7accumulated in the cells is about 70 mg/100 g of dry cells, which isequivalent to about 80 times the amount (870 μg/100 g) in the nattoshown in the table of food product analysis published by Science andTechnology Agency. Incidentally, the amounts of MK-7, MK-4, and vitaminK₁ accumulated in the microorganic cells indicated in the presentspecification are numerical values determined by the method which isdescribed in detail as below.

[0047] When the cultured product of Bacillus subtilis produced by themethod of this invention is collected by the known method such asfiltration or centrifugal separation and then dried by the well-knownmethod such as freeze drying, air drying, and vacuum heat drying and thedry cells are dissolved in water, the vitamin K is dissolved out in thewater. From this fact in combination with the fact that the cellscontain a large amount of vitamin K (particularly MK-7), it may beassumed that the vitamin K (particularly the MK-7) accumulated in thecells becomes water-soluble because it has undergone some sort of changein the cells. The vitamin K, vitamin K₂, and menaquinone-7 in the formthus endowed with this water-solubility will be referred to in thepresent specification as “water-soluble vitamin K derivative” (or simplyas “vitamin K derivative”,}, “water-soluble vitamin K₂ derivative” (orsimply as “vitamin K₂ derivative”), and “water-soluble menaquinone-7”(or water-soluble MK-7 derivative) respectively. In view of the factthat the menaquinone-7 has a molecular weight of about 649 incombination with the fact that the water-soluble menaquinone-7derivative exhibits a single band of a molecular weight of about 100,000when measured by the SDS-polyacrylamide gel electrophoresis and amolecular weight of not less than 100,000 when measured by the gelfiltration, it can be suspected that the water-soluble vitamin Kderivative (inclusive of a vitamin K₂ derivative and a menaquinone-7derivative; which stipulation will be omitted hereinafter) is presentinside the cells in such a state that vitamin K including vitamin K₂ andmenaquinone-7 binds a certain substance (such as, for example,glycopeptide) and stabilized. It goes without saying, however, that thepostulate just mentioned will not limit the concept of this invention.

[0048] In this invention, the vitamin K derivative, vitamin K₂derivative, and menaquinone-7 derivative have the solubility in watervaried with the species of the microorganism, the kind of the culturemedium to be used, the culturing conditions, the method for treating(extracting) the cultured product. For example, the water-solubility ofthe vitamin K derivative, in the case of extracting cultured cells withwater, is about 150 μg/100 ml water (20° C), in the case of thesupernatant of the cultured medium, is about 300 μg/100 ml water (20°C.), and in the case of extracting cultured natto with water, is about1,500 μg/100 ml water (20° C.).

[0049] According to the fourth aspect, this invention provides a foodproduct, beverage, or feed containing the cultured product according tothe second aspect and/or the water-soluble vitamin K derivativeaccording to the third aspect as mentioned above.

[0050] The food product, beverage, or feed according to this invention,particularly when the microorganism to be used has the safety thereofestablished as in the case of Bacillus subtilis natto, may be composedsolely of the cultured product according to the second aspect and/or thewater-soluble vitamin K derivative according to the third aspect asmentioned above.

[0051] Alternatively, the food product, beverage, or feed according tothis invention may have the cultured product according to the secondaspect and/or the water-soluble vitamin K derivative according to thethird aspect as mentioned above incorporated in a food product,beverage, or feed which can be normally consumed by eating. As typicalexamples of the food product, beverage, or feed intended for normalconsumption by eating, bean curd refuse, fermented bean curd refuse,natto, soybean, soybean soup stock by-produced during the manufacture ofsoybean paste and fermented soybeans (natto), soybean curd leesby-produced during the manufacture of soybean curd (tofu) and deep-friedsoybean curd, soybean lees by-produced during the manufacture of edibleoil from soybean as the raw material, soybean seed rind by-producedduring the manufacture of soybean paste, dairy products such as yogurtand cheese, kneaded marine products such as boiled fish paste,tube-shaped fish paste cake, cake of pounded fish, bar-shaped fishpaste, and fish ball, processed seafood such as mashed and seasonedfish, processed meat products such as sausage, frankfurter, and leverpaste, soybean products such as soybean curd (tofu), broiled soybeancurd, fried bean curd, deep-fried soybean curd, fried bean curd cakehaving vegetables and other ingredients wrapped therein, bean curdrefuse, freeze dried bean curd, and sheet of bean curd, processedvegetable products such as puree, processed potato products such asmashed potato, arrowroot starch, bean-starch vermicelli, konjak, andnoodles made from devil's tongue starch, processed cereal products suchas rice cake, rice-flour dumplings, boiled rice, breadlike food made ofwheat gluten, rice vermicelli, macaroni, spaghetti, fine noodles,buckwheat noodles, noodles, Chinese noodles, instant noodle, loaf ofbread, ship biscuit, and bun filled with bean jam, frozen food,nutritional supplementary food, sweeteners such as jam, oils and fatssuch as butter, margarine, mayonnaise, and dressing, confectioneryproducts such as candy, molded dry cake, very small rice biscuits,sponge cake, sweet jellied adzuki-bean paste, bean-jam-filled wafers,bun filled with bean jam, soft rice cake filled with bean jam, dumpling,sweetened cake, chocolate, biscuit, cookies, doughnut, cakes, pies, icecream, pudding, bavarois, and chewing gum, gel-like food products suchas bean curd, jelly, konjak, agar, and gelidium jelly, marine plantssuch as kelp, seaweed, laver, and agar weed, and all the other foodproducts; various fruit juices (orange, pineapple, apple, grape, melon,and strawberry), various bicarbonate beverages, tea (inclusive of greentea and oolong tea), drinking yoghurt, milk, soy milk, modified milk,mineral water, soft drink, coffee, black tea, cocoa, and all the otherbeverages; and feed for normally kept animals such as domestic animalslike pig, cow, horse, sheep, and goat, pets such as dog, cat, rabbit,and hamster, and poultry and fish may be cited.

[0052] When the food product, beverage, or feed contains the culturedproduct according to this invention, the amount of the cultured productin the food product, though variable with the species of microorganismto be used, the kind of culture medium, the culturing conditions, andthe content of water-soluble vitamin K derivative, is such that the foodproduct generally contains 0.001 to 20% by weight of dry cells,preferably 0.1 to 5% by weight of dry cells, the beverage generallycontains 0.0001 to 5 (w/v)% of dry cells, preferably 0.01 to 5 (w/v)% ofdry cells, and the feed generally contains 0.0001 to 5% by weight of drycells, preferably 0.001 to 1% by weight of dry cells. When the foodproduct, beverage, or feed contains the water-soluble vitamin Kderivative according to this invention, the amount of the water-solublevitamin K derivative in the food product, though variable with thespecies of microorganism to be used, the kind of culture medium, and theculturing conditions similarly in the case mentioned above, is such thatthe food product generally contains 0.00001 to 10% by weight ofwater-soluble vitamin K derivative, preferably 0.0001 to 0.1% by weightof water-soluble vitamin K derivative, the beverage generally contains0.00001 to 0.1 (w/v)% of water-soluble vitamin K derivative, preferably0.0001 to 0.01 (w/v)% of water-soluble vitamin K derivative, and thefeed generally contains 0.00001 to 10% by weight of water-solublevitamin K derivative, preferably 0.0001 to 1% by weight of water-solublevitamin K derivative.

[0053] By ingesting the food product, beverage, or feed containing thecultured cells containing the water-soluble vitamin K (particularlyMK-7) derivative of this invention in a large amount and/or thewater-soluble vitamin K (particularly MK-7) derivative originating inthe cultured cells, therefore, the vitamin K, particularly the MK-7 canbe taken efficiently in a large amount at a time without any unduestress (without entailing any undue discomfort) as compared with theconventional vitamin K. When the microorganism is Bacillus subtilisnatto, this food product proves advantageous because of establishedsafety.

[0054] According to the fifth aspect, this invention provides a methodfor extracting a vitamin K which comprises Soxhlet-extracting thecultured cells of Bacillus subtilis.

[0055] One embodiment of this aspect is described as below. Firstly,cultured microorganic cells are Soxhlet-extracted with such an organicsolvent as hexane, diethylether, acetone, ethanol and isopropanol byusing a Soxhlet extractor at a boiling temperature of the used solventto obtain a fat-soluble fraction. Then, this fraction is extracted fromsuch an organic solvent as hexane, diethylether, acetone, ethanol andisopropanol at 30° to 100° C. for 0.1 to 20 hours. The resultant extractis diluted with the same organic solvent as just used to a prescribedtotal volume. An aliquot of the diluted extract is intimately mixed withwater and isopropanol and further mixed with the same organic solvent asjust used as by the use of a touch mixer. The resultant liquid mixtureis centrifuged and the supernatant is dried to hardness and the dryresidue is dissolved in ethanol to prepare a fat-soluble vitamin Kincluding vitamin K₂ (MK-7, MK-4), and vitamin K₁ having a higherpurity.

[0056] By this method, a fat-soluble vitamin K can be extracted moreefficiently as compared with the conventional methods and thus afat-soluble vitamin K including vitamin K₂ (MK-7, MK-4), and vitamin K₁can be recovered from the cultured cells with a higher yield.

[0057] According to sixth aspect, this invention provides a method forfractioning a water-soluble vitamin K derivative which comprisesacidifying a culture medium of Bacillus subtilis to obtain aprecipitate.

[0058] One embodiment of this aspect is described as below. Bacillussubtilis cells are cultured by a conventional method or the method asdescribed above to obtain a culture medium. Then, the pH of this culturemedium is decreased, preferably to a level in the range of 1 to 3 toform a precipitate which contains a water-soluble vitamin K derivativeto be accumulated in the cells and to be released out of the cells. Bysubjecting this precipitate to separation as by centrifugal separation,a water-soluble vitamin K derivative can be obtained.

[0059] Alternatively, Bacillus subtilis cells are cultured by aconventional method or the method as described above to obtain a culturemedium. The resultant culture medium is separated as by centrifugalseparation to obtain a supernatant. Then, the pH of this supernatant isdecreased, preferably to a level in the range of 1 to 3 to form aprecipitate which contains a water-soluble vitamin K derivative to bereleased out of the cells. By subjecting this precipitate to separationas by centrifugal separation, a water-soluble vitamin K derivative canbe obtained.

[0060] Now, this invention will be described more specifically belowwith reference to working examples.

[0061] The amounts of MK-7, MK-4, and vitamin K₁ mentioned in thefollowing examples were measured according to the following methods.

[0062] <Method for Measuring Amounts of Vitamin K's>

[0063] First, the method for preparing sample cells of fat-soluble MK-7,MK-4, and vitamin K₁ will be described below. 1 g (dry weight) ofcultured microorganic cells as a sample are extracted with a Soxhletextractor (SIBATA SPC 34, WATER BATH SIBATA WB-6C, filter paper ADVANTEC84 24×100 mm) to obtain a fat-soluble fraction. This fraction isextracted from 100 ml of hexane at 80° C. for 6 hours. The resultantextract is diluted with hexane to a total volume of 100 ml. The extract,100 μl in volume, is intimately mixed with 1.0 ml of distilled water and1.5 ml of isopropanol and further mixed with 4.9 ml of hexane by using atouch mixer for about 10 seconds. The resultant mixture is centrifuged(3,000 rpm×10 minutes, 20° C.). A 4.0 ml portion of the supernatant(organic layer:aqueous layer=5.8:1.7) is concentrated and dried tohardness by evaporation and the residue is dissolved in 100 μl ofethanol. As a result, an HPLC sample for measuring the amounts offat-soluble MK-7, MK-,4, and vitamin K₁ accumulated in the cells(hereinafter referred to as “Soxhlet-HPLC sample”) is prepared. Themethod for measuring the amount of MK-7 by the following HPLC using thisSoxhlet-HPLC sample will be referred to as “Soxhlet-HPLC method”.

[0064] Then, the method for preparing a sample of the water-solublevitamin K derivative (inclusive of MK-7) will be described herein below.A cultured product is thoroughly kneaded by means of a spatula. To 5 gof the kneaded cells, 45 ml of distilled water is added. The resultantmixture is centrifuged at 3,000 rpm at 20° C. for 10 minutes. Thesupernatant, 0.5 ml in volume, is used as an extraction sample. To thisextraction sample, 0.5 ml of distilled water and 1.5 ml of isopropanolare added and intimately mixed, and then 5.0 ml of hexane is furtheradded. The resultant mixture is stirred and then centrifuged at 3,000rpm at 20° C. for 10 minutes. The resultant supernatant, 4.0 ml involume, is concentrated and dried to hardness by the use of anevaporator and the residue is dissolved in 100 μl of ethanol. Thus, anHPLC sample for measuring the amount of water-soluble MK-7 derivativeaccumulated in the cells (hereinafter referred to as “HPLC sample”) isprepared. The method for measuring the amount of MK-7 by the followingHPLC using this HPLC sample will be referred to as “HPLC method”.

[0065] As these HPLC samples for measuring the amounts of MK-7, MK-4,and vitamin K₁, the relevant cultured broth is used in its unmodifiedform.

[0066] The measurement of the amounts of MK-7, MK-4, and vitamin K₁ bythe high-performance liquid chromatography (HPLC) makes use of thephenomenon that vitamin K is reduced by a platinum-alumina catalyst intoa hydroquinone compound and made to form a fluorescent substance. To bemore specific, this measurement is carried out under the followingconditions. <Apparatus> Pump: PU-980 (made by Nippon Bunko K.K.)Injector: 7125 (made by Nippon Bunko K.K.) Column Oven: CO-960 (made byNippon Bunko K.K.) Detector: Fluorescent Detector 821-FP (made by NipponBunko K.K.) Data Processing: C-R5A (made by Shimadzu Seisakusho DeviceLtd.) <Conditions> Column: ODS-II column (4.6 × 250 mm) (made byShimadzu Seisakusho Ltd.) Reduction Column: Platinum-Alumina CatalystColumn (measuring 4.0 mm in diameter and 10 mm in length, made by WakoPure Chemical Industries Ltd., packed with about 0.2 g ofplatinum-alumina catalyst of first grade having a Pt content of 5%)Mobile Phase: 97% ethanol (flow rate: 0.7 ml/min.) Separation ·Reduction: 40° C. Temperature Measuring Wavelength: Excitation 320 nmand fluorescence 430 nm Amount Injected: 10 μl

[0067] When calibration curves for MK-7, MK-4, and vitamin K₁ wereprepared in accordance with the method of measurement described above,the amount of MK-7 could be measured in the range of 0.05 to 50 ng bythe formula [−0.89661+1.6993×10⁻⁶ 33 (area of the HPLC corresponding tovitamin K (μV, sec))] and the amounts of MK-4 and vitamin K₁ could bemeasured in the range of 0.01 to 10 ng respectively by the formula[−0.58657+4.8030×10⁻⁹×(area of the HPLC corresponding to vitamin K (μV,sec) )] and the formula [−0.44381+4.0626×10⁻⁷×(area of the HPLCcorresponding to vitamin K (μV, sec))].

[0068] The standard specimen of MK-7 used in the formation of thecalibration curves was prepared as follows. Specifically, 600 g offermented soybeans (natto) and 1 liter of 75% isopropanol and 1 liter ofn-hexane added thereto were slowly stirred for one hour and the producedmixture was left standing. The upper layer of the two separated layerswas removed, dried with anhydrous sodium sulfate, and evaporated tohardness to obtain about 20 g of an extract. This extract was mixed with10 ml of n-hexane. The solution was passed for adsorption through 400 mlof a bed of chromatography grade silica gel. The adsorbate was elutedand fractionated with 2 liters of n-hexane/toluene (1:1) mixture. Thefractions containing MK-7 were evaporated to dryness under a reducedpressure. The combined silica gel concentrate was dissolved in 5 ml ofn-hexane. The resultant solution was fractionated with the silica gelcolumn in the same manner as described above and the fraction wasevaporated to dryness under a reduced pressure to obtain a residue,about 350 mg in weight. A 50 mg portion of the residue was dissolved ina small amount of acetone. The resultant solution was passed through a60 ml column of chromatography grade ODS-silica gel packed withacetonitrile/methanol (1:1) and developed therein withacetonitrile/methanol (1:1). Wile the eluate was monitored by the HPLC,the fraction having eluted solely therein a substance suspected to beMK-7 was separated and dried to hardness under a reduced pressure. Theinfrared absorption spectrum and the mass spectrum obtained of thissubstance identified this substance to be MK-7. This substance wastested for purity, to find to have a purity of 99.8%. The standardspecimens of phylloquinone (vitamin K₁) and menaquinone-4 (MK-4) usedherein were relevant reagent grade products of Sigma Corp. The resultsof the measurement were obtained by averaging the analyses of threeextractions.

EXAMPLE 1

[0069] 10 culture media, each 200 ml in volume, (total amount of 2liters) was prepared by dissolving dry bouillon (made by Nissui SeiyakuK.K.) in a concentration of 3% in 0.3% malt soup stock in 5 Erlenmeyerflasks having an inner volume of 500 ml, and then sterilized in anautoclave at 130° C. for about 30 minutes. After the sterilized solutionwas cooled, about 5×10⁸ cells of Miyagino strain (made by Miyagino NattoManufactory K.K. in Sendai) was placed in this solution and thenshaking-cultured (100 rpm) at 40° C. for 36 hours. The cultured cellswere collected, washed, mixed with a substantially equal amount of cornstarch, and air dried at 4° C. over a period of three days. The driedmixture consequently obtained contained a large amount of live cells ofBacillus subtilis natto (spores and vegetative cells). When it wastested for amount of menaquinone-7 (MK-7) accumulated therein by theHPLC method, it was found to have a menaquinone-7 content of about 331.7μg per g of dry cells.

[0070] Then, five healthy adults were each caused to ingest 1 g of thedry cells at 10 o'clock every morning. After the elapse of a prescribedinterval, blood was collected from each of the healthy adults and wastested for MK-7 level in the plasma. The time-course changes of MK-7level in the plasma were investigated. The results are shown in FIG. 1.It is noted from FIG. 1 that clear acceleration of the level in plasmawas recognized and this acceleration was found to last for not less than24 hours (p<0.05).

EXAMPLE 2

[0071] Five healthy adults were each caused to ingest 1 g of dry cells(containing 1.8×10¹⁰ live cells) of Nitto strain (made by Nitto YakuhinKogyo K.K. in Kyoto) at 10 o'clock every morning. Then, blood wascollected from the adults along the course of time. The plasma wasseparated from the collected blood and tested for MK-7 level in theplasma in the same manner as in Example 1. The results are shown inTable 1 and FIG. 2. The Bacillus subtilis natto used in the presentexample had a MK-7 content, as determined by the HPLC method, of about51.0 μg/g of dry cells.

[0072] As a control, five healthy adults were caused to ingest purifiedMK-7 in place of the dry cells of Bacillus subtilis natto mentionedabove. The plasma similarly obtained from each of the healthy adults wastested for MK-7 level in the plasma. The results are shown in Table 1below. TABLE 1 Concentration of MK-7 in plasma (after the elapse of) 0hour 4 hours 8 hours 12 hours 24 hours 32 hours 48 hours B. subtilisnatto 1.2 ± 1.1  7.5 ± 2.7** 5.0 ± 1.0** 4.8 ± 1.3** 4.3 ± 1.9* 4.8 ±0.9** 3.1 ± 0.6* (about 1.8 × 10¹⁰ cells/g) Purified MK-7 1.3 ± 1.1 11.2± 2.8** 7.3 ± 1.6** 4.3 ± 1.0** 2.2 ± 1.0  1.4 ± 0.7†  1.4 ± 0.9† (about51.0 μg)

[0073] It is noted from the results shown in Table 1 and FIG. 2 that asignificant acceleration of the MK-7 level in plasma reaching the peakon the fourth hour after the ingestion was recognized (p<0.005)invariably in both the groups, that the effect of accelerating the levelin plasma lasted for a far longer time in the group of adults havingingested Bacillus subtilis natto, and that particularly the effect inand after the 32nd hour had a significant difference between that of thegroup of adults having -ingested MK-7 and that of the group of adultshaving pure MK-7.

[0074] With this amount of ingestion, none of the groups was recognizedto bring any significant change in the activity of the bloodcoagulation-fibrinolysis system as investigated by thethromboelastography, the activated partial thromboplastin time, theplasma prothrombin, or the protein C content. Incidentally, thethromboelastography (TEG) pattern of the blood was investigated with thedevice made by Hellige Corp. and the prothrombin time (PT) and theactivated thromboplastic time (APTT) were determined with the instrumentmade by Erma Corp. and sold under trademark designation of Clot DigitimTE20 apparatus.

EXAMPLE 3

[0075] The following six species of Bacillus subtilis natto were eachshaking-cultured (100 rpm) at 30° C. in 300 ml of a culture mediumcontaining 3% nutrient broth (dry bouillon) (made by Nippon SeiyakuK.K.) and held in an Erlenmeyer flask, 500 ml in inner volume. Thegrowth of the microorganic cells was determined in terms of absorbanceat 660 nm and the amounts of MK-7 in the cultured broth and in the cellswere measured at prescribed periods.

[0076] The growth of each species of Bacillus subtilis natto, the amountof MK-7 in the cultured broth (μg/ml), and the amount of MK-7 per gramof the cells of each species of Bacillus subtilis natto (μg/g)respectively relative to the culturing time are shown in FIG. 3, FIG. 4,and FIG. 5, respectively.

[0077] <Bacillus subtilis Natto Used>

[0078] Unnan SL-001 Strain(otherwise called simply as “Unnan”)

[0079] Miyagino strain (made by Miyagino Natto Manufactory K. K. inSendai)

[0080] Meguro strain (made by Meguro Laboratory K.K. in Osaka)

[0081] Nitto strain (made by Nitto Yakuhin Kogyo K.K. in Kyoto)

[0082] Takahashi strain (made by Yuzo Takahashi Laboratory in Sendai)

[0083] Naruse strain (made by Naruse Fermentation Chemical Laboratory K.K. in Tokyo)

[0084] Hereinafter, the aforementioned Bacillus subtilis natto will bereferred to by names of species.

[0085] It is noted from FIG. 3 that the microorganic growth showed asimilar behavior in all the species of Bacillus subtilis natto and thegrowth expressed in terms of average absorbance at 660 nm (n=6) was0.583 on the 0.5th day, 1.011 on the first day, 0.948 on the second day,0.854 on the third day, 0.592 on the fifth day, and 0.552 on the seventhday, and that the microorganic growth reached the stationary state onthe first-second day after start of the culture. As respects the amountof MK-7 released into the culture broth, it is noted from FIG. 4 thatall the species of Bacillus subtilis natto manifested a similar behaviorand the average amount for the six species of Bacillus subtilis nattowas 0.563 μg/ml on the 0.5th day, 1.592 μg/ml on the first day, 3.867μg/ml on the third day, 4.317 μg/ml on the fifth day, and 4.784 μg/ml onthe seventh day. These facts indicate that the amount of MK-7 releasedinto the culture medium abruptly increased rather on and after thesecond day than on the first-second day on which the microorganic growthreached the stationary state and that the MK-7 was released in a largeamount into the culture medium particularly on and after the fourth day.The amount of MK-7 reduced to the amount of MK-7 per g of themicroorganic cells (similarly all the species of Bacillus subtilis nattoexhibited a similar behavior), as shown in FIG. 5, reached the maximum(about 300 μg/g of dry cells on the average) on the second day andrather began to decrease on and after the fourth day on which therelease in a large amount into the culture medium started, as thoughproportionally to the results mentioned above.

EXAMPLE 4

[0086] Bean-curd refuse (made by Asahimatsu Shokuhin K.K. in Iida-shi)was stored in a frozen state at −25° C. and, when necessary, defrostedand put to use. The defrosted bean curd refuse was sterilized in anautoclave at 120° C. for 30 minutes and placed in a container ofpolystyrene paper (PSP) having an inner volume of about 120 ml and usedas a culture medium for Bacillus subtilis natto.

[0087] A total of seven species of Bacillus subtilis natto, i.e. fourstrains of Bacillus subtilis natto (Takahashi strain, Naruse strain,Miyagino strain, and Asahi strain) used in commercially availablefermented soybeans, two strains of Bacillus subtilis natto (Nitto strainand Merugo) used in medicines, and a strain of Bacillus subtilis nattoseparated from the fermented soybeans from Unnan Province of China(Unnan SL-001 strain), were each shaking-cultured (100 rpm) at 37° C.for three days in 150 ml of a culture medium containing 3% nutrientbroth (made by Nippon Seiyaku K.K.) and held in an Erlenmeyer flask, 500ml in inner volume to prepare pre-cultured broths of the seven speciesof Bacillus subtilis natto mentioned above.

[0088] 0.5 ml of the pre-cultured broths of the total of seven speciesof Bacillus subtilis natto (live cell number: 2×10⁸ cells/ml) were eachadded to the bean-curd refuse prepared as described above (50 g of wetweight) and left standing at 37° C. for eight days to undergo continuedfermentation.

[0089] After the elapse of prescribed numbers of days (1, 2, 4, 6, and 8days) after start of the fermentation, the Bacillus subtilis natto wassuspended in water and the suspension was filtered through a teastrainer made of metal and then centrifuged (3,000 rpm×10 minutes) toprepare relevant cultured microorganic cells. These cultured cells wereused as Soxhlet-HPLC samples for the measurement of MK-7. TheSoxhlet-HPLC samples thus prepared were tested for the amount of MK-7and the fibrinolytic activity (thrombolysis). The results are shown inFIG. 6.

[0090] It is noted from FIG. 6 that the level of MK-7 in themicroorganic cells per 10¹⁰ cells increased on the first-second day anddecreased thereafter, that the release of MK-7 into the culture mediumproceeded proportionately to the behavior of the concentration, and thatthe amount of MK-7 released into the culture medium reached the peak onthe fourth day which was later than the time at which the thrombolyticactivity by nattokinase (NK) reaching the peak on the second day afterstart of the fermentation began to increase (the activity reaching themaximum within two days of starting the fermentation by the standardfibrin plate method and the synthetic substrate decomposition methodalike).

[0091] In all the species of Bacillus subtilis natto, such high levelsof MK-7 productivity as exceed 1.9 μg per g of the wet weight of beancurd refuse were recognized (the average amount of MK-7 dissolved inwater was 36.6 μg/g of the wet weight of bean curd refuse in Unnan; 1.9μg/g of the wet weight of bean curd refuse in Miyagino strain; 14.2 μg/gof the wet weight of bean curd refuse in Naruse strain; 6.8 μg/g of thewet weight of bean curd refuse in Takahashi strain; 11.9 μg/g of theweight of bean curd refuse in Asahi strain; 1.9 μg/g of the wet weightof bean curd refuse in Meguro strain; and 5.2 μg/g of the weight of beancurd refuse in Nitto strain). Particularly, Unnan showed such a highlevel of MK-7 productivity as 36.6 μg/g of the wet weight of bean curdrefuse. This value is shown by calculation to be so high as 2 to 20times the level of productivity obtained by other species of Bacillussubtilis natto and so high as not less than four times the levels ofanalyses of fermented soybeans (6.2 to 8.7 μg/g of the wet weight ofbean curd refuse) reported heretofore [Table of Japanese Food ProductComponents, complied by Michio Yamaguchi, published by Ishiyaku Shuppan,Tokyo, 1997, pp. 52-53; Toshiyuki Sakano et al., Vitamins, 62, 393-398(1988); H. Ikeda and Y. Doi, Eur. J. Biochem., 192, 219-223 (1990); andH. Ikeda, Kaseishi, 43, 643-648 (1992)].

EXAMPLE 5

[0092] Nitto strain was added in such an amount to 300 ml of a liquidculture medium containing 0.5, 2, or 3% of polypeptone S (made by WakoPure Chemical Industries Ltd.) and held in an Erlenmeyer flask, 500 mlin inner volume that the culture medium contained 2×10⁶ livemicroorganic cells per ml and shaking-cultured (100 rpm) therein at 37°C. for two days. The resultant culture broth was centrifuged (3,000rpm×10 minutes) to separate a supernatant and microorganic cells. Thecells were washed with water and freeze dried.

[0093] The amount of MK-7 in the culture supernatant thus prepared wasmeasured by the HPLC method and the amount of MK-7 in the microorganiccells was measured by the Soxhlet-HPLC method. The results are shown inTable 2 below. The yield of microorganic cells in the culture mediahaving polypeptone S concentrations of 0.5 and 3% were respectively 0.24g (weight of dry cells) and 1.18 g (weight of dry cells) per 600 ml ofculture medium (for two flasks). TABLE 2 Amount of MK-7 in Amount ofMK-7 in Polypeptone S supernatant microorganic cells Concentration(μg/ml) (μg/g of dry cells) 0.5% 1.11  73.4   2% 0.62 140.0   3% 0.51245.3

EXAMPLE 6

[0094] Meguro strain was shaking-cultured at 41° C. for two days. Theculture broth consequently obtained was centrifuged to separate asupernatant and microorganic cells.

[0095] Then, an aliquot, 1.011 g (dry weight), of the microorganic cells(number of cells: 5×10¹¹) thus prepared was adopted as a sample andextracted by the use of a Soxhlet extractor (SIBATA SPC 34, WATER BATHSIBATA WB-6C, filter paper ADVANTEC 84 24×100 mm) to obtain afat-soluble fraction over a period of 6 hours. This fraction wasextracted from 100 ml of hexane at 80° C. for 6 hours. The liquidextract was diluted with hexane to a total volume of 100 ml. Theextract, 100 μl in volume, was intimately mixed with 1.0 ml of distilledwater and 1.5 ml of isopropanol and further mixed with 4.9 ml of hexaneby the use of a touch mixer for about 10 seconds. The resultant mixturewas centrifuged (3,000 rpm×10 minutes, 20° C.). The produced supernatant(organic layer:water layer=5.8:1.7), 14 ml in volume, was dried tohardness and the dry residue was dissolved in 100 μl of ethanol. Whenthe sample thus obtained was tested for the MK-7 content by theSoxhlet-HPLC method, the content was found to be 672.6 μg/g of drycells.

EXAMPLE 7

[0096] Meguro strain was cultured at 37° C. for two days which periodcan make the largest amount of a vitamin K derivative accumulated withinthe cells. The cultured product thus obtained was washed with distilledwater and then freeze-dried. Five healthy adults (A to E) were eachcaused to ingest an enteric capsule in which 1 g of freeze-dried product(containing 5×10¹¹ cells/g of dried product) was filled. In this case,the MK-7 content was determined by the Soxhlet-HPLC method method, tofind to be 708.0 μg/g of dried product. At prescribed points, blood wascollected from the adults along the course of time. The plasma MK-7level was tested in the same manner as in Example 1. The results areshown in Table 3. TABLE 3 Plasma MK-7 Level at Prescribed Points afterHealthy Administration of Enteric Capsule (ng/ml) Adult 0 Day 0.5 Day 1Day 3 Days 7 Days A 1.1 9.3 8.9 4.5 2.4 B 1.0 10.6  7.4 5.1 2.2 C 0.99.1 8.2 2.6 1.9 D 0.6 6.8 6.9 2.3 1.4 E 0.8 8.7 6.6 2.6 1.7 Average 0.88± 8.90 ± 7.60 ± 0.95* 3.42 ± 1.28* 1.92 ± 0.40* 0.19 1.37*

[0097] The results shown in Table 3 indicates a significant accelerationof the MK-7 level in plasma. At the same time, as shown in Table 3, theMK-7 level in plasma was kept at a level of about 2 times as high as thenormal plasma MK-7 level (0.88±0.19 ng/ml) for at least one week andthus this acceleration continues over an extremely long period.

EXAMPLE 8

[0098] The MK-7 prepared from Meguro strain in the same manner as inExample 6 was dried to obtain drain MK-7 in the form of white powder.This dry MK-7 was exposed to the light from a fluorescent lamp at roomtemperature for 5 hours. In this case, the amount of MK-7 was measuredalong the course of time in the same manner as in Example 6. The resultsare shown in FIG. 7 (indicated by empty circle (◯) marks). The amount ofMK-7 showed virtually no change on exposure to the light from thefluorescent lamp as noted from FIG. 7, indicating that the MK-7 obtainedby the method of the present invention manifests significantly excellentstability to withstand the light (photostability).

[0099] When the dry MK-7 prepared as described above was exposedcontinuously to the light of the fluorescent lamp for one week under thesame conditions as mentioned above and then tested for the MK-7 content,virtually no change was recognized.

COMPARATIVE EXAMPLE 1

[0100] Microorganic cells were prepared in the same manner as in Example6. In a mortar, 2 g of the cells were placed and 3 g of sea sand and 3ml of water were added to the cells in the mortar. Then, the content ofthe mortar was stirred and extracted with acetone (20 ml×4) and suctionfiltered by the use of a glass filter. The filtrate was whollytransferred into a separation funnel and extracted from 100 ml ofdiethyl ether and 50 ml of water to separate an ether layer. The aqueouslayer was combined with 100 ml of diethyl ether for further separationof the ether layer. The ether layers thus separated were combined,distilled to expel the solvent by evaporation, and subjected to silicagel chromatography (hexane 10 ml and silica gel 5 g). The adsorbate onthe silica gel was eluted with 30 ml of hexane/diethyl ether [85/15(v/v)]. The eluate was further distilled to expel the solvent byevaporation. The residue was dissolved in 2-propanol to prepare afat-soluble MK-7.

[0101] The fat-soluble MK-7 thus prepared was dried to obtain a dryproduct of MK-7. This dry product of MK-7 was irradiated with the lightfrom a fluorescent lamp at room temperature for 5 hours and then testedfor the fat-soluble MK-7 content in the same manner as in Example 6. Theresults are shown in FIG. 7 (indicated by filled square (▪) marks). Theamount of MK-7 could not be measured after the sample was exposed to thelight from the fluorescent lamp for about 3 hours as noted from FIG. 7,indicating that the MK-7 thus prepared lacks in photostability.

EXAMPLE 9

[0102] Nitto strain was shaking-cultured and the resultant culture brothwas centrifuged to separate a supernatant and microorganic cells. Whenthe amount of MK-7 in the cultured microorganic cells thus prepared wasmeasured by the Soxhlet-HPLC method, the content was found to be 564.0μg/g of dry cells, in terms of MK-7 amount. 0.1 g of aliquot of thecells and 5 ml of distilled water added thereto were subjected tosonication. Then, the sonicated liquid was centrifuged. When the amountof the water-soluble MK-7 derivative in the supernatant was measured bythe HPLC method, the content was found to be 52.6 μg/g of dry cells.

[0103] Then, a solution of the water-soluble MK-7 derivative in waterwas subjected to gel filtration by the use of a Sephadex G50 column(2.5×50 cm: equilibrated in advance with a 0.05M phosphate buffercontaining 0. 15M NaCl, pH 7.5). The content of MK-7 in each fractionwas measured by the HPLC method. The results are shown in FIG. 8. It isnoted from FIG. 8 that the portions containing MK-7 in large amounts(Fraction Nos. 17 to 25) were eluted in fairly higher molecular weightregions than the amino acid portions (Fraction Nos. 46 to 49).

EXAMPLE 10

[0104] The bean curd refuse offered by Asahimatsu Shokuhin K.K. inIida-shi was stored in a frozen state at −25° C. and, when necessary,defrosted and put to use.

[0105] Separately, a pre-cultured broth of Bacillus subtilis natto wasprepared by shaking-culturing (100 rpm) a strain of Bacillus subtilisnatto separated from fermented soybeans imported from Unnan Province ofChina (Unnan SL-110 Strain) at 37° C. for 3 days in 150 ml of a culturemedium containing 3% nutrient broth (dry bouillon) (made by NissuiSeiyaku K.K.) in an Erlenmeyer flask having an inner volume of 500 ml.

[0106] The defrosted bean curd refuse, 1 kg in wet weight, wassterilized in an autoclave at 120° C. for 30 minutes and then placed ina container of polystyrene paper (PSP). In this container, thepre-cultured broth of Bacillus subtilis natto prepared as describedabove was placed and left fermenting at 37° C. for 4 days. The bean curdrefuse, 1 kg in wet weight which had been fermented as described aboveand 5 liters of water added thereto were stirred at room temperature forone hour and centrifuged (3,000 rpm×10 minutes). The supernatant and 620g of an ion-exchange resin (DEAE-Sepharose CL-613) added thereto werestirred and then left standing at room temperature for 30 minutes. Then,the resultant mixture was filled in a glass column (7.5 cm in diameter X100 cm), washed with distilled water and 0.05M phosphate buffer (pH7.0), and subjected to gradient elution with 0.05M phosphate buffer (pH7.0) containing 0.1 to 0.8 M NaCl. The amounts of vitamin K weremeasured by the HPLC. The fraction containing vitamin K was concentratedby the use of a membrane filter (molecular weight 10,000, made byMillipore Corp.), dialyzed against distilled water, and thenfreeze-dried to obtain a water-soluble vitamin K derivative in the formof a light yellow powder.

[0107] The water-soluble vitamin K derivative obtained as describedabove and the extract of the same bean cure refuse as mentioned abovefrom isopropanol were analyzed by the HPLC. The results are shown inFIG. 9. In FIG. 9, the intervals of retention of MK-4, vitamin K₁, andMK7 were respectively about 8.2 minutes, about 11.3 minutes, and about17.5 minutes. The arrow mark indicates the retention time of MK-7. It isnoted from FIG. 9 that a portion of not less than 60% of the extract ofbean curd refuse from isopropanol was extracted in the water-solublefraction, that the amounts of vitamin K₁ and MK-4 were small as vitaminK, and that a portion of not less than 95% was always occupied by MK-7.

[0108] When the water-soluble vitamin K derivative was analyzed by theSDS-polyacrylamide gel electrophoresis, it showed a sole band, thoughlarge in width, at the position of a molecular weight of about 100,000.From the results, it is inferred that the water-soluble vitamin Kderivative forms a complex of a molecular weight of about 100,000 withother glycoprotein. The yield of water-soluble vitamin K derivative from1 kg of fermented soybeans was about 5.3 g (inclusive of 830 μg ofwater-soluble MK-7 derivative/g), an average of the results of threeprocedures.

EXAMPLE 11

[0109] Erlenmeyer flasks (an inner volume of 500 ml) each having 300 mlof 3% dry bouillon (made by Nissui Seiyaku K.K.) media placed thereinwere sterilized in an autoclave at 120° C. for 15 minutes. In thesemedia, each one loop of Unnan SL-001 strain was inoculated andshaking-cultured (100 rpm) at 37° C. After 4 days, the combined mediumwas centrifuged (5,000 rpm×10 minutes), and the supernatant was dividedinto three so as to make each the volume thereof 400 ml. Then, the pHvalue of each aliquot was adjusted to 1.02, 2.07 and 3.01 with dilutehydrochloric acid. These aliquots were left standing at a roomtemperature for three hours and then centrifuged (5,000 rpm×10 minutes,at 10° C.), to obtain and separate a white precipitate. Each precipitatewas dissolved in a small amount of distilled water, made the pH valueadjusted to 7.0 with ammonium bicarbonate, and thereafter freeze-dried.The freeze-dried products were weighed to find to be 0.52 g, 0.28 g and0.31 g, in the case of the pH of 1.02, 2.07 and 3.01, respectively.Further, these freeze-dried products were tested for the MK-7 contentsby the Soxhlet-HPLC method to find that the MK-7 contents in the case ofthe pH of 1.02, 2.07 and 3.01 were 2,800 μg/g dry product, 2,200 μg/gdry product and 2,000 μg/g dry product, respectively. Furthermore,solutions having 15 mg of each freeze-dried product added to 5 ml ofdistilled water were centrifuged (10,000 rpm×10 minutes). Thesupernatants (i.e. water-soluble fractions) were similarly tested forthe MK-7 content to find that the MK-7 contents were 1,500 μg/g dryproduct, 1,800 μg/g dry product and 1,800 μg/g dry product, in the caseof the pH of 1.02, 2.07 and 3.01, respectively, indicating that thesolubility of each water-soluble fraction was about 54%, about 82% andabout 90%, respectively.

EXAMPLE 12

[0110] 3.9 kg of Soybean which had been immersed in tap water overnightwas sterilized in an autoclave at 105° C. for 30 minutes, and thenspread in two stainless trays of 1 m² in size. To these trays, 100 ml ofa sterilized water having added in 100 ml of sterilized water 10 loopsof Bacillus subtilis Warburgt (Oyo Biseibutsu Kenkyusho of TokyoUniversity, IAM 12118 strain) which was cultured on a slant of anutrient agar medium was inoculated. A lid of urethane was placed oneach tray and stationary-cultured at 37° C. for 4 days. After 4 days,the combined weight of fermented soybean (of 2 trays) was about 4.5 kg.Then, 10 liters of distilled water was added to this fermented soybean,stirred intimately, and filtered through a metal gauze covered withgauze (mesh of 10 mm). The water-soluble MK-7 content of the resultantfiltrate was determined by the HPLC method to find that total amount(i.e., per 4.5 kg fermented soybean (wet weight)) of 29.8 mg/g could berecovered.

EXAMPLE 13

[0111] 300 ml of a culture medium containing 2% of polypeptone-S (madeby Wako Pure Chemical Industries Ltd.) was placed in an Erlenmeyerflask, 500 ml in inner volume and sterilized in an autoclave at 120° C.for 15 minutes. To this medium, one loop of Bacillus subtilis Warburgt(Oyo Biseibutsu Kenkyusho of Tokyo University, IAM 12118 strain) wasinoculated and shaking-cultured (100 rpm) therein at 37° C. for 4 days.The cultured medium was centrifuged (6,000 rpm×10 minutes) to separate asupernatant and microorganic cells. The cells were washed with water andfreeze dried.

[0112] The supernatant was tested by the HPLC method for the MK-7content to find to be 140 μg/100 ml. Further, the freeze dried cellswere tested by the Soxhlet-HPLC method for the MK-7 content to find tobe 22 mg/100 g dried cell.

EXAMPLE 14

[0113] Five healthy adults (21 to 63 years old, male) requested toabstain from breakfast were each made to ingest 1, 000 Rg of thewater-soluble vitamin K derivative containing the water-soluble MK-7derivative prepared in Example 9 at 10 o'clock in the morning. Blood wascollected from the healthy adults along the course of time after theingestion and measured for the level of the water-soluble MK-7derivative in plasma. As a control, the experiment was repeated with theingestion of 1,000 μg of purified MK-7 instead. The healthy adults whoparticipated in the experiment of this example had no past illness andshowed no abnormality in the plasma test.

[0114] It is found from the results that the main vitamin in plasma wasMK-7, that the level of the water-soluble MK-7 derivative in plasmawhich was 1.3±0.8 ng/ml (plasma) before the ingestion rose to 49.9±29.1ng/ml (p<0.05), a value about 40 times the initial value, on the fourthhour after the ingestion, and that the effect of acceleration thereoflasted for far greater lengths of time than that attained by theingestion of purified MK-7 in the same amount (on the 24th hour, thelevel was 21.0±12.1 ng/ml, p<0.05, in the group of the water-solubleMK-7 derivative, while the level was 3.2±2.3 ng/ml in the group ofpurified MK-7).

EXAMPLE 14

[0115] In a kettle, 400 g of milk, 80 g of sugar, and 8 g of corn starchwere mixed by stirring and meanwhile heated. The mixture was cooled withthe fire turned down immediately before the content of the kettleboiled. The mixed liquid thus cooled and 10 g of a freeze dried culturedproduct of Miyagino strain produced in the same manner as in Example 3(730 μg of vitamin K/g of dry cells) added thereto were thoroughlystirred for about two minutes. Separately, in a bowl, 40 g of egg yolkwas whipped. The whipped egg yolk and the stirred mixture mentionedabove added wholly thereto gradually were mixed. Subsequently, theproduced mixture was cooled with water and then cooled to below 10° C.in a refrigerator. Finally, the cooled mixture and 150 g of whippedfresh cream and 5 g of vanilla essence added thereto were cooled tohardness in an ice cream freezer to manufacture a vitamin K-containingice cream.

EXAMPLE 15

[0116] In a jar fermenter, 2 liters in inner volume, Nitto strain wasinoculated to 0.8 liter of a culture medium (containing 10 g of glucose,5 g of polypeptone, and 5 g of yeast extract dissolved in 1 liter ofwater) and fermented at 40° C. for 1.5 days by aeration culture bydiffusion (stirring rate of 500 rpm and aeration rate of 0.5liter/minute).

[0117] The amount of MK-7 in the cultured product of Nitto strainobtained as described above was measured by the conventional method(Sakano et al., Vitamins, 62: 393-398, 1988). Briefly, the measurementwas carried out by freeze-drying the cultured product of Nitto strainobtained as described above, causing 0.1 g of the freeze-dried powderand 10 ml of isopropanol added thereto to be stirred and extracted in atouch mixer, and then centrifuging the resultant mixture (3,000 rpm×10minutes). 100 μl of portion of the supernatant was intimately mixed with1.0 ml of water and 1.5 ml of isopropanol and further mixed with 4.9 mlof hexane for about 20 seconds in a touch mixer. The resultant mixturewas centrifuged (3,000 rpm×10 minutes, at 20° C.). A 4.0 ml portion ofthe supernatant (organic layer: water layer=5.8: 1.7) was concentratedand dried to hardness by evaporation and dissolved in 100 μl of ethanol.When the sample thus prepared was measured for the MK-7 content in thecultured product under the same conditions as the HPLC conditionsdescribed in the preceding paragraph concerning the method for measuringthe amounts of various species of vitamin K, the amount of MK-7 wasfound to be 15.1 μg/g. When the procedure just described was repeated byusing a chloroform/methanol (1:1) mixture and ether respectively inplace of isopropanol, the amount of MK-7 was found to be 15.0 μg/g and13.6 μg/g, respectively. These amounts were nearly equal to the amountof MK-7 obtained when isopropanol was used.

[0118] In contrast, the cultured product of Nitto strain obtained in thesame manner as described above was measured for the amount of MK-7 bythe Soxhlet-HPLC method. As a result, the amount of MK-7 in the culturedproduct of Nitto strain was found to be 564.0 μg/g.

[0119] These results indicate that the Soxhlet-HPLC method according tothe present invention improved the efficiency of extraction of MK-7 andmeasured satisfactorily the MK-7 content in the cultured product, whilethe conventional method allowed efficient extraction of the microorganiccells of Bacillus subtilis natto only with difficulty and failed tomeasure the MK-7 content in the cultured product satisfactorily.

[0120] The entire disclosure of Japanese Patent Application Nos.10-172,019 and 11-111,364 filed on May 17, 1998 and Apr. 19, 1999including specification, claims, drawings and summary are incorporatedherein by reference in its entirety.

1. A method for culturing Bacillus subtilis which comprises the steps ofculturing Bacillus subtilis and recovering the cultured microorganiccells before the vitamin K produced within the cells is released fromsaid cells.
 2. A method according to claim 1, wherein said microorganiccells are recovered at the time when the number of cells is in theprocess of shifting from the logarithmic growth phase to the maximumstationary phase.
 3. A method according to claim 1 or claim 2, whereinsaid Bacillus subtilis is Bacillus subtilis natto.
 4. A method accordingto claim 3, wherein said microorganic cells are recovered during theperiod in which the number of cells is in the process of shifting fromthe logarithmic growth phase to the maximum stationary phase and beforenattokinase is produced.
 5. The cultured product of Bacillus subtiliscultured by the method set forth in any of claims 1 to
 4. 6. Awater-soluble vitamin K derivative originating in the cultured productof Bacillus subtilis set forth in claim
 5. 7. A vitamin K derivativeaccording to claim 6, wherein said vitamin K derivative is vitamin K₂derivative.
 8. A vitamin K derivative according to claim 7, wherein saidvitamin K₂ derivative is menaquinone-7 derivative.
 9. A food product,beverage, or feed containing the cultured product set forth in claim 5and/or the water-soluble vitamin K derivative set forth in any of claims6 to
 8. 10. A method for extracting vitamin K which comprisesSoxhlet-extracting cultured cells of Bacillus subtilis.
 11. A method forfractioning a water-soluble vitamin K derivative which comprisesacidifying a culture medium of Bacillus subtilis to obtain aprecipitate.